Alpha beta T cells respond to peptide-major histocompatibility complex (MHC) molecule ligands. How protein antigens are transformed into peptides suitable for such binding and the intracellular pathways followed by MHC molecules both before and after peptide association are critical to understanding T cell immunity. MHC class II molecules assemble in the presence of invariant chain (Ii) and Ii plays important roles in regulating the early trafficking of MHC class II, as well as directing it movement in the endocytic pathway. We have found that association of the ER-resident protein calnexin with Ii prevents Ii degra-dation, and retains it in this organelle, so that it achieves a high molar ratio to class II and saturates class II binding sites in the endoplasmic reticulum. New studies show that interaction of Ii varies with the allele of class II. This appears to reflect binding site polymorphism affecting association with the CLIP seg-ment of Ii. Other regions of Ii modulate this CLIP-related variation in binding strength, in accord with our modular model of Ii. Using transfected cells expressing various combinations of wild-type and mutant MHC class II and Ii molecules, we have found that Ii is essential for presentation of only some peptide determinants within a single protein antigen. Signals in the cytoplasmic tails of the MHC class II alpha and beta chains control Ii-independent presentation of other determinants. These data suggest that two separate pools of MHC class II molecules (newly synthesized and recycling, mature) provide for maximal effec-tive capture of antigenic information. Pulse-chase labelling, immunoprecipitation, and gradient density fractionation of B lymphoblasts show that class II traffics to multiple endocytic compartments, consistent with class II acquisition of different protein determinants in compartments of differing pH and hydrolytic capacity. Intact extracellular proteins are usually not converted into peptides bound to MHC class I mole-cules, but some exceptions involving particulate antigen have been reported. We have examined the ability of phagocytic stimuli to promote class I presentation of exogenous soluble protein. Our studies suggest that this class I presentation under these conditions reflects the rare breakdown of the phagosome membrane and entry of the antigen into the conventional cytoplasmic class I processing pathway. These data are of significance for vaccine design, and may also bear on CD8 T cells responses to some pathogens that normally reside in endocytic vesicles, but occasionally escape into the cytoplasm.